WO2002050293A2 - Plants with improved resistance - Google Patents

Abstract

The invention relates to transgenic plants with improved resistance to pathogens, whereby the improved resistance is a result of an increased expression of one or more nucleic acid sequences according to SEQ ID NO:1 to 11, or homologues, fragments or derivatives thereof, or by a change in the biological activity of the genetic product coded by one or more nucleic acid sequences according to SEQ ID NO:1 to 11, or the homologues, derivatives or fragments thereof. The invention further relates to transgenic plants with at least one regulatory nucleic acid sequence according to SEQ ID NO:12 to 23, integrated into the genome in a stable manner and a nucleic acid sequence coding for a genetic product functionally linked to the above nucleic acid sequence. The invention furthermore relates to a method for the production of the said transgenic plants and the nucleic acid sequence according to SEQ ID NO:1 to 23.

Description

 Plants with improved resistance

The present invention relates to transgenic plants with improved resistance to pathogens, the improved resistance by increased expression of one or more nucleic acid sequences according to SEQ LD NO: 1 to 11, or their homologs or derivatives or fragments, or by changing the biological activity of one or more nucleic acid sequences according to SEQ LD NO: 1 to 11, or their homologs or derivatives or fragments encoded gene products. The present invention further relates to transgenic plants with at least one regulatory nucleic acid sequence according to SEQ LD NO: 12 to 23 which is stably integrated into the genome, and a nucleic acid sequence which is functionally linked to a gene product and which is functionally linked to this nucleic acid sequence. The present invention further relates to methods for producing the transgenic plants according to the invention and the nucleic acid sequences according to SEQ LD NO: 1 to 23.

The attack of certain phytopathogens induces a wide range of defense reactions in infected host plants. Common defense strategies include, for example, the rapid death of plant cells at the site of infection (hypersensitive response), structural cell wall reinforcement through the incorporation of polymeric components around the site of penetration, the production of hydrolytic antipathogenic enzymes and the synthesis of phytoalexins with antimicrobial activity. At the beginning of an infection, specific molecular pathogen components, so-called elicitors, are usually bound by plant receptors, which informs the plant about the infection and initiates countermeasures that include numerous enzymatic processes.

The generic term WRKY genes is used to summarize a number of genes that code for structurally related putative transcription factors in plants. The members of the WR Y family regulate a wide variety of plant physiological processes such as B. Pathogen defense, wound healing, trichoma development and senescence. So far, however, very few WRKY proteins have known the specific metabolic processes in which they are involved intervention. The current state of WRKY research is at T. Eulgem, PJ. Rushton, S. Robatzek and IE Somssich (2000): The WRKY superfamily of plant transcription factors, Trends in Plant Sciences 5, 199-206. The WRKY protein superfamily in the "thale cress" Arabidopsis thaliana currently comprises about 70 different transcription factors, which together have an approximately 60 amino acid DNA-binding WRKY domain (with the amino acid sequence WRKYG (QK)). The DNA binding will mediated by a zinc finger motif of this domain.

Due to the number of WRKY domains and the structure of zinc cells, WRKY proteins are classified into three groups. Group I proteins contain two, group II proteins only one WRKY domain. Group III WRKYs also have only one WRKY domain, but differ from the members of Group II by a different zinc finger motif (type C ₂ -HC). It is not possible to infer the physiological function of the WRKYs based on the group membership.

To date, there are more than 500 WRKY ESTs from various plant species in databases. Many are tissue specific and have been isolated from roots, leaves, flowers or seeds or are only used under certain conditions, e.g. B. salt or dry stress expressed. At the moment, only some of these WRKY genes have specific information about which metabolic pathway they are involved in. Few have been shown to be involved in defense against plant pathogens: WRKYl and WRKY 3 from Petroselinum crispum (Eulgem, T. et al: Early nuclear events in plant defense signaling: rapid gene activation by WRKY transcription factors. EMBO Journal 18, 4689-4699, 1999) and WRKY3 and WRKY4 from Nicotiana tabacum (Chen, C. and Chen, Z: Isolation and characterization of two pathogen- and salicylic acid induced genes encoding WRKY DNA-binding proteins from tobacco. Plant Molecular Biology 42, 387 -396, 2000). A pathogen defense function of the WRKY proteins 30, 33, 41, 46, 54, 55, 62, 63, 64, 67 and 70 mentioned in this patent application was not previously known.

The task of WRKY proteins in the defense against pathogens presumably consists in the transcriptional activation of special genes. So z. B. in cell suspension cultures the induction of the PR1-1 and PR1-2 genes of Petroselinum crispum by elicitors secreted by the fungus, inter alia by a defined fungal oligopeptide (Pep25). The Binding of Pep25 to a plant plasma membrane receptor leads to an increase in various ion concentrations, to phosphorylation / dephosphorylation of numerous proteins and ultimately to the activation of genes involved in the defense against pathogens, which also include PR1-1 and PR1-2. The promoters of these genes contain so-called W boxes (W1, W2 and W3) with the sequence (T) (T) TGAC (C / T), which act as binding sites for sequence-specific DNA-binding WRKY proteins. The WRKY proteins thus represent transcription factors for genes, the promoters of which contain some copies of the W boxes. Genes whose promoters have W-boxes can e.g. B. for pathogen-damaging proteins such as antimicrobial chitinases or glucanases. The promoters of most WRKY genes also contain W boxes. These genes are consequently regulated by other WRKY genes.

Almost all modern crops are infected by pathogenic organisms and sometimes severely damaged. These pathogens include various fungi, bacteria, nematodes and viruses. The plants have different natural defense mechanisms, but they often only provide limited protection against the pathogens. For this reason, the intensive use of arable land in modern agriculture and horticulture requires the frequent use of chemical pesticides, if they are available. Regardless of all pesticides and methods developed so far, as well as the successful classic resistance breeding in crops, this is nowadays Infestation with pathogens resulting crop loss is still significant and causes worldwide economic damage of several billion marks annually. The development of new plants with improved resistance or a wider range of resistance could help to reduce the problems mentioned.

The present invention is therefore based on the object of providing novel pathogen-resistant plants and processes for their production.

The object is achieved by the subject-matter defined in the patent claims.

The invention is illustrated by the following figures.

Figure 1 shows the genomic nucleic acid sequence of the WRKY30 gene from Arabidopsis thaliana. The start ATG and stop codon are highlighted in bold. Underlined regions represent introns.

FIG. 2 shows the genomic nucleic acid sequence of the WRKY33 gene from Arabidopsis thaliana. The start ATG and stop codon are highlighted in bold. Underlined regions represent introns.

FIG. 3 shows the genomic nucleic acid sequence of the WRKY41 gene from Arabidopsis thaliana. The start ATG and stop codon are highlighted in bold. Underlined regions represent introns.

FIG. 4 shows the genomic nucleic acid sequence of the WRKY46 gene from Arabidopsis thaliana. The start ATG and stop codon are highlighted in bold. Underlined regions represent introns.

FIG. 5 shows the genomic nucleic acid sequence of the WRKY54 gene from Arabidopsis thaliana. The start ATG and stop codon are highlighted in bold. Underlined regions represent introns.

FIG. 6 shows the genomic nucleic acid sequence of the WRKY55 gene from Arabidopsis thaliana. The start ATG and stop codon are highlighted in bold. Underlined regions represent introns.

FIG. 7 shows the genomic nucleic acid sequence of the WRKY62 gene from Arabidopsis thaliana. The start ATG and stop codon are highlighted in bold. Underlined regions represent introns.

FIG. 8 shows the genomic nucleic acid sequence of the WRKY63 gene from Arabidopsis thaliana. The start ATG and stop codon are highlighted in bold. Underlined regions represent introns.

FIG. 9 shows the genomic nucleic acid sequence of the WRKY64 gene from Arabidopsis thaliana. The start ATG and stop codon are highlighted in bold. Underlined regions represent introns.

FIG. 10 shows the genomic nucleic acid sequence of the WRKY67 gene from Arabidopsis thaliana. The start ATG and stop codon are highlighted in bold. Underlined regions represent introns.

FIG. 11 shows the genomic nucleic acid sequence of the WRKY70 gene from Arabidopsis thaliana. The start ATG and stop codon are highlighted in bold. Underlined regions represent introns.

Figure 12 shows the promoter region of the Arabidopsis thaliana WRKY33 gene. The 1270 bp promoter fragment (SEQ ID NO: 12) according to FIG. 21 is highlighted in italics and the 361 bp promoter fragment (SEQ LD NO: 13) according to FIG. 21 is underlined twice. The forward primers for the 1270 bp, 361 bp, 331 bp and 193 bp fragment according to FIG. 21 and the reverse primer of the four fragments are highlighted in bold. The start ATG is in the reverse primer. Lower case letters denote the coding region of the WRKY33 gene in the reverse primer.

FIG. 13 shows an RT-PCR analysis for detecting the induction of the WRKY genes 30, 41, 46, 62, 63, 64 and 67 after infection of Arabidopsis thaliana with the leaf pathogen Peronospora parasitica pv Cala2. Samples were taken at the time of infection (0) as well as 2, 4, 6, 12 and

24 hours after infection (hpi = hours post induction). To prove that the

Induction caused by the pathogen infections were triggered

Control infections with pure water (H ₂ 0) carried out instead of a pathogen application.

FIG. 14 shows an RT-PCR analysis for the detection of the induction of the WRKY genes 54, 55 and

70 after infection of Arabidopsis thaliana with the leaf pathogen Peronospora parasitica pv

Cala2. Gene-specific primers were used for the analysis. Sampling took place on

Time of infection (0) and 2, 4, 6, 12 and 24 hours after infection (hpi = hours post induction). To demonstrate that the induction was caused by the pathogen infections, control infections with pure water (H ₂ 0) instead of one

Pathogen application carried out.

FIG. 15 shows a Northern blot for the detection of the induction of WRKY 33 after infection of Arabidopsis thaliana with the leaf pathogen Peronospora parasiticum parasitica pv Cala2. Samples were taken at the time of infection (0) and 2, 4, 6 and 24 hours after the infection (hpi = hours post induction). To prove that the induction was caused by the pathogen infections, control infections with pure water (H ₂ 0) were carried out instead of a pathogen application.

FIG. 16 shows the local induction of WRKY33 by means of GUS staining of Peronospora parasitica-mΑzi & sXQXi (right) and uninfected Arabidopsis thaliana plants (left). Part A shows GUS-colored primary leaves, part B corresponding cotyledons. Staining was carried out for 2 hours 2 days after infection.

FIG. 17 shows the local induction of WRKY33 by means of GUS staining of Arabidopsis thaliana plants infected with Alternaria α / terπαtα. Part A shows an overall view of the mycelium infection on Arabidopsis leaves, part B shows a 2.5-fold enlargement of an infection site. Admission was made 9 days after infection.

FIG. 18 shows the local induction of WRKY33 by means of GUS staining of Sclerotina sclerotiorum-efficient Arabidopsis thaliana plants. Part A shows an overall view of the mycelium infection on Arabidopsis leaves two or three days after the infection, part B shows the 5-fold enlargement of an infection site. Admission was made 9 days after infection.

FIG. 19 shows the induction of WRKY33 by means of GUS staining of Arabidopsis thaliana plants after contact with the root pathogen Pythium sylvaticum. Part A shows an overall view of uninfected plants (control), Part B plants that were infected with Pythium sylvaticum. The partial images C and D represent detailed images of the root area of infected plants. All images were taken two weeks after the infection.

FIG. 20 shows a tabular list of the transcription factor binding sites which occur in the promoters of the Arabidopsis thaliana WRKY genes 33, 41, 46, 54, 55, 62, 63, 64, 67 and 70. For the investigation, 1.5 kilobase-long promoter sections before the respective transcription start point were taken into account and these were searched with computer support for known transcription factor binding sites. FIG. 21 shows a schematic representation of the promoter analysis carried out according to Example 3. Constructs consisting of WRKY33 promoter portions of different lengths and the beta-glucoronidase reporter gene (uidA) were used for expression studies. A strong induction was found with a 1270 bp promoter portion, while 361 bp was sufficient for a basal induction. No induction was achieved with 331 and 193 long promoter parts.

FIG. 22 shows a Northern blot to demonstrate that WRKY33 is located in the RPP2-dependent signal chain and that its expression is dependent on a functional PAD4 protein. Samples were taken at the time of infection (0) and 2, 4, 6, 24, 48 and 72 hours after the infection (hpi = hours post induction).

The terms “homologs” or “homologous sequences” used here denote non-Arabidopsis thaliana-derived nucleic acid or amino acid sequences with significant similarity to the comparison sequence or parts thereof and identical physiological function in other plants. Homologous sequences are therefore nucleic acid sequences with identical physiological function in other plants which hybridize with the comparison sequences or parts of these sequences under stringent conditions (for stringent conditions see Sambrook et al, Molecular Cloning, Cold Spring Harbor Laboratory (1989), ISBN 0-87969 -309-6). An example of stringent hybridization conditions is: Hybridization in 4 x SSC at 65 ° C (alternatively in 50% formamide and 4 X SSC at 42 ° C), followed by several washing steps in 0.1 x SSC at 65 ° C for a total of about one Hour. Furthermore, homologous sequences are to be considered nucleic acid or amino acid sequences or parts thereof with identical physiological function in other plants, which with the aid of the BLAST similarity algorithm (Basic Local Alignment Search Tool, Altschul et al, Journal of Molecular Biology 215, 403-410 (1990 ) have a significant similarity to comparison sequences. Sequences are described as significantly similar, as used here, which are used, for example, using standard parameters in the BLAST service of the NCBI (http://www.ncbi.nlm.nih.gov/ BLAST /) have a level of significance (E-Value or Probability) of P <10 ^{* 5} when compared with the comparison sequences.

The term "homologs" or "homologous sequences" used here denotes the further nucleic acid or amino acid sequences or parts thereof, which are at least 70%, preferably 80%, particularly preferably 90% and particularly preferably 95%) identical to the comparison sequences. The deviations from the comparison sequences may have resulted from deletion, substitution, lisertion, inversion or recombination.

The term “derivatives” used here denotes nucleic acids which also carry the genetic information for the protein encoded by the comparison sequence, although their base sequence differs from that of the comparison sequence. In this sense, the term “derivatives” denotes both genomic equivalents of RNA containing introns, EST or cDNA sequences as well as equivalents of the comparison sequence that have arisen due to the degeneration of the genetic code.

The term "fragments" used here denotes parts of nucleic acid or amino acid sequences, provided that they have at least one functionally important region (domain, sequence or structural motif) of the comparison sequence. Corresponding functionally important regions are, in particular, elements of DNA binding, eg . (1) a WRKY- protein domain that includes the consensus sequence WRKYG, (2) zinc-finger motifs with the consensus sequences CX ₄ CX _{5. 22} - ₂₃ -HX ₁ -H or CX ₂ -CX 3-HX ₁ -C (3 ) Structural elements that contribute to binding to so-called W-boxes with the base sequence (T) (T) TGAC (C / T) Further functionally important areas are binding sites for transcription factors, catalytic centers, amino acid motifs interacting with proteins, eg receptor or ligand binding sites, as well as phosphorylation, acetylation or myristylation sites.

The term "expression" used here means the transcriptional transcription of genetic information in RNA and, in the case of protein-coding genes, the subsequent translation into polypeptides.

The expression "enhanced expression" or "enhanced gene expression" used here means an increase in the amount of transcript formed to more than 100% in comparison with the corresponding amount of transcript in wild type plants.

The term "endogenous" used here denotes biological components such as. B. Gene sequences that come naturally from the very organism in which they are or should be integrated.

The term "exogenous" used here denotes biological components such as. B. gene sequences that naturally originate from a different organism in which they are integrated or are to be integrated.

The term "functionally linked" used here means that a regulatory sequence such as a promoter controls the expression of a gene or another functional nucleic acid or that a nucleic acid sequence is expressed starting from the promoter.

The term "functional nucleic acid" or "functional nucleic acid sequence" used here means a nucleic acid sequence which does not code for a naturally occurring gene product, e.g. B. a ribozyme, an antisense DNA or RNA or a sequence composed of different exons.

The term "vector" used here denotes naturally occurring or artificially created constructs for the uptake, multiplication, expression or transfer of nucleic acids, e.g. B. plasmids, phagemids, cosmids, artificial chromosomes, bacteriophages, viruses, retroviruses.

The term “transgenic plant” used here relates to plants which were produced by means of recombinant genetic engineering and / or microbiological processes and not by means of conventional breeding processes.

The term "expression system" used here denotes any combination of vectors, restriction enzymes, transformation methods, cell extracts, living cells, for. B. prokaryotic or eukaryotic cells or organisms with the purpose of expression of genes.

It was surprisingly found on the basis of infection experiments with various phytopathogens that a number of Arabidopsis thaliana were found in the thale cress of WRKY genes with hitherto completely unexplained physiological function can be induced by infestation of the plant with a pathogen. Induction begins shortly after contact with the pathogen and is essentially local to the site of the infection. In particular, a corresponding pathogen-induced activation of the genes coding for WRKY30, WRKY33, WRKY41, WRKY54, WRKY55, WRKY62, WRKY63, WRKY64, WRKY67 and WRKY70 was detected. It was found that the induction is not limited to a pathogen, but can be triggered by various pathogens.

The present invention thus relates to transgenic plants with one or more nucleic acid sequence (s) stably integrated into the genome according to SEQ LD NO: 1, SEQ TD NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ TD NO: 5 , SEQ ID NO: 6, SEQ TD NO: 7, SEQ LD NO: 8, SEQ LD NO: 9, SEQ ID NO: 10 or SEQ ID NO: II, or their homologue or derivative or fragment, or a nucleic acid sequence with an identical one physiological function which hybridizes with one of these nucleic acid sequences, preferably hybridizes under stringent conditions, and leads to the development of an improved resistance to pathogens in the plants concerned, and methods for the production of the transgenic plants.

The above-mentioned SEQ FD NO: 1 to 11 can be assigned to the WRKY genes described in this application according to the following list:

SEQ ID NO: 1 corresponds to the sequence of the gene coding for WRKY30.

SEQ ID NO: 2 corresponds to the sequence of the gene coding for WRKY33.

SEQ TD NO: 3 corresponds to the sequence of the gene coding for WRKY41.

SEQ TD NO: 4 corresponds to the sequence of the gene coding for WRKY46. SEQ ID NO: 5 corresponds to the sequence of the gene coding for WRKY54.

SEQ TD NO: 6 corresponds to the sequence of the gene coding for WRKY55.

SEQ TD NO: 7 corresponds to the sequence of the gene coding for WRKY62.

SEQ TD NO: 8 corresponds to the sequence of the gene coding for WRKY63.

SEQ TD NO: 9 corresponds to the sequence of the gene coding for WRKY64. SEQ ID NO: 10 corresponds to the sequence of the gene coding for WRKY67.

SEQ ID NO: 11 corresponds to the sequence of the gene coding for WRKY70.

An improvement in the resistance to pathogens is, for example, through a to achieve one or more of the following methods:

(a) an increased expression of one or more endogenously present WRKY genes,

(b) an introduction and expression of additional WRKY gene copies into the plant genome, (c) a change in the biological activity of WRKY gene products.

In case groups a) and b), a larger or larger amount of WRKY proteins is formed intracellularly as a result of the expression which is temporarily or constitutively increased in comparison to wild-type plants. In the further course of the reaction chain, the WRKY transcription factors interact with promoter elements (so-called W-boxes), which are located in front of genes that are relevant to pathogen defense (so-called “defense related genes”) and increase their transcription by binding. By means of a higher cellular concentration of WRKY proteins more secondary products of this metabolic pathway are also formed and the immune defense of the plant is significantly improved.Transgenic plants which, due to increased gene expression or additional WRKY gene copies, produce an increased amount of WRKY transcripts or their fragments or homologues or derivatives compared to the wild type show one significantly improved pathogen defense.

An increased expression can accordingly z. B. by combining the nucleic acid sequence according to SEQ LD NO: 1, SEQ TD NO: 2, SEQ TD NO: 3, SEQ ID NO: 4, SEQ LD NO: 5, SEQ LD NO: 6, SEQ ID NO: 7, SEQ LD NO: 8, SEQ ID NO: 9, SEQ TD NO: 10 or SEQ ID NO: 11 or their homologue or derivative or fragment can be achieved with a strong promoter. Examples of suitable promoters which can lead to an increased constitutive expression of a WRKY gene or its homolog or fragment or derivative are the CaMV 35S promoter from the cauliflower mosaic virus and the ubiquitin promoter from maize. For this purpose, either the endogenous promoter which expresses one of the nucleic acid sequences according to SEQ ID NO: 1, SEQ ID NO: 2, SEQ LD NO: 3, SEQ LD NO: 4, SEQ ID NO: 5, SEQ LD NO: 6, SEQ ID NO: 7, SEQ LD NO: 8, SEQ TD NO: 9, SEQ ID NO: 10 or SEQ LD NO: l 1 or their homolog or derivative or fragment controls, can be replaced by the strong promoter, or at least an additional copy of one of the nucleic acid sequences according to SEQ TD NO: 1, SEQ ID NO: 2, SEQ LD NO: 3, SEQ LD NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ TD NO: 7, SEQ LD NO: 8, SEQ TD NO: 9, SEQ ID NO: 10 or SEQ ID NO: 11 or their homolog or derivative or fragment integrated into the genome with the additional ^) copy (s) being (are) functionally linked to the strong promoter.

Enhanced gene expression of the nucleic acid sequences according to SEQ LD NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ LD NO: 4, SEQ LD NO: 5, SEQ LD NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ LD NO: 9, SEQ LD NO: 10 or SEQ LD NO: l 1 or their fragment or homolog or derivative can also be achieved by changing or adding functional components of the promoter which naturally regulates the expression of the WRKY gene become. Essential components of a typical eukaryotic promoter are state of the art and can be found in relevant textbooks. Examples of corresponding functional components are TATA box, CAAT box, GC box, enhancer or binding sites for other transcription factors. Suitable methods for changing the nucleic acid sequence of the promoter which naturally regulates the expression of the WRKY gene are known to the person skilled in the art. Known mutagenesis techniques are, for example, the generation of deletion mutants or the introduction of point mutations by "site directed mutagenesis". Preferred methods for mutagenesis or for changing the expression are furthermore chimeric RNA / DNA oligonucleotides, homologous recombination or RNA interference (RNAi).

Enhanced expression can also be achieved by more than one additional copy of the nucleic acid sequences according to SEQ LD NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ TD NO: 5, SEQ ID NO : 6, SEQ LD NO: 7, SEQ ID NO: 8, SEQ TD NO: 9, SEQ LD NO: 10 or SEQ ID NO: 11 or their homologue or derivative or fragment, in particular two, three, four, five or more Copies to be integrated into the genome. These copies can be integrated individually or contiguously. If more than one of the nucleic acid sequences listed is integrated, several identical copies or a mixture of the nucleic acid sequences mentioned can be used. Furthermore, the copies are functionally linked to a promoter which is a strong promoter, an endogenous or an exogenous one, the expression of the nucleic acid sequences according to SEQ LD NO: 1, SEQ LD NO: 2, SEQ LD NO: 3, SEQ TD NO: 4, SEQ LD NO: 5, SEQ TD NO: 6, SEQ LD NO: 7, SEQ LD NO: 8, SEQ LD NO: 9, SEQ ID NO: 10 or SEQ ID NO: II or their homolog or derivative-controlling promoter, or can be a tissue-specific or inducible promoter. Constructs comprising a corresponding promoter and a nucleic acid sequence functionally linked to it according to SEQ ID NO: 1, SEQ LD NO: 2, SEQ ID NO: 3, SEQ LD NO: 4, SEQ LD NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ LD NO: 8, SEQ ID NO: 9, SEQ LD NO: 10 or SEQ LD NO: II or their homologue or derivative or fragment can be converted by conventional methods for the transformation of plant cells and subsequently for the regeneration of complete plants be used. The introduction of nucleic acid sequences into plant organisms and cells is state of the art and can be carried out easily, for example using T-DNA vectors. Due to the increased expression of the nucleic acid sequences according to SEQ LD NO: 1, SEQ ID NO: 2, SEQ LD NO: 3, SEQ TD NO: 4, SEQ ID NO: 5, SEQ LD NO: 6, SEQ TD NO: 7, SEQ TD NO: 8, SEQ TD NO: 9, SEQ LD NO: 10 or SEQ LD NO.T 1 or their homologue or derivative or fragment are accordingly formed a multiple of proteins encoded by these nucleic acid sequences.

According to case group c) there is a change in the biological activity of the nucleic acid sequences according to SEQ TD NO: 1, SEQ LD NO: 2, SEQ LD NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ LD NO: 6 , SEQ ID NO: 7, SEQ LD NO: 8, SEQ LD NO: 9, SEQ LD NO: 10 or SEQ ID NO: 11 or their homolog or derivative or fragment-encoded gene product by changes in gene segments which are necessary for functional protein domains such as e.g. , B. encode receptor binding sites, phosphorylation domains or DNA binding sites. Modifications in the WRKY DNA binding domain in particular have a considerable influence on the properties of the protein. In this way, proteins can be formed with one or more amino acids which have been changed compared to the wild type and which, for example, have a changed substrate specificity or a changed K _m value or are no longer subject to the regulatory mechanisms normally present in the cell via allosteric regulation or covalent modification. Correspondingly modified WRKY proteins could accordingly. B. perform the functions of the WRKY wild type protein in an improved manner. In this sense, in particular modifications which result in the replacement of one or more of the following amino acids by others, the loss of one or more of the following amino acids or the integration of one or more amino acids in the following protein regions can lead to a correspondingly improved function of the protein :

WRKY 30 GVDRT DDGFSWRKYGQKDI GAKFPRGYYRCTYRKSQGCEATKQVQRSDENQMLLEISYRGIHSCSQ (SEQ ID NO: 24) WRKY 33 SDIDILDDGYRWRKYGQKWKGNPNPRSYYKCTTIGCP KHVERASHDMRAVITTYEGKHNHDVPAA

(SEQ ID NO: 25)

WRKY 41 GLEGPHDDIFSWRKYGQKDILGAKFPRSYYRCTFRNTQYCWATKQVQRSDGDPTIFEVTYRGTHTCSQ

(SEQ ID NO: 26) WRKY 46 QENGSIDDGHCWRKYGQKEIHGSKNPRAYYRCTHRFTQDCLAVKQVQKSDTDPSLFEVKYLGNHTCNN

(SEQ ID NO: 27)

WRKY 54 VEAKSSΞDRYAWRKYGQKEI NTTFPRSYFRCTHKPTQGCKATKQVQKQDQDSEMFQITYIGYHTCTA

(SEQ ID NO: 28)

WRKY 55 NTDLPPDDNHTWRKYGQKEILGSRFPRAYYRCTHQKLYNCPAKKQVQRLNDDPFTFRVTYRGSHTCYN (SEQ ID NO: 29)

WRKY 62 SRTMCPNDGFTWRKYGQKTIKASAHKRCYYRCTYAKDQNCNATKRVQKIKDNPPVYRTTYLGKHVCKA.

(SEQ ID NO: 30)

WRKY 63 SPNPRLDDGFTWRKYGQKTIKTS YQRCYYRCAYAKDQNCYATKRVQMIQDSPPVYRTTYLGQHTC A

(SEQ ID NO: 31) WRKY 64 SPTPRPDDGFTWRKYGQKTIKTSPYQRCYYRCTYAKDQNCNARKRVQMIQDNPPVYRTTYLGKHVCKA

(SEQ ID NO: 32)

WRKY 67 SSTPIYHDGFLWRKYGQKQIKESEYQRSYYKCAYTKDQNCEAKKQVQKIQHNPPLYSTTYFGQHICQL

(SEQ ID NO: 33)

WRKY 70 IESTILEDAFSWRKYGQKEILNAKFPRSYFRCTHKYTQGCKATKQVQKVE EPKMFSITYIGNHTCNT (SEQ ID NO: 34)

Suitable methods for changing the sequence of the WRKY gene are known to the person skilled in the art. Known mutagenesis techniques are, for example, the generation of deletion mutants or the introduction of point mutations by "site directed mutagenesis". Preferred methods for mutagenesis or for changing the expression are furthermore chimeric RNA / DNA oligonucleotides, homologous recombination or RNA interference (RNAi). Transgenic plants with such modified WRKY genes also have an improved resistance to pathogens.

The nucleic acid sequences used in the method according to the invention and in transgenic plants can be of natural origin or have been produced artificially.

Nucleic acid sequences with an identical physiological function to the nucleic acid sequences according to SEQ ID NO: 1, SEQ TD NO: 2, SEQ ID NO: 3, SEQ TD NO: 4, SEQ TD NO: 5, SEQ TD

NO: 6, SEQ TD NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ LD NO: 10 or SEQ ID NO: ll can be easily extracted from other organisms with the help of special, state of the art PCR, hybridization or screening procedures are isolated. For example, the nucleic acid sequences listed above can be used as a probe for homology screening in DNA libraries with the aid of hybridization to single-stranded nucleic acids of a similar base sequence. Furthermore, by knowing the base sequence of the nucleic acid sequences listed above, primers can be constructed which can be used to amplify functionally identical PCR fragments from other organisms.

Those with the nucleic acid sequences according to SEQ ID NO: 1, SEQ ID NO: 2, SEQ TD NO: 3, SEQ ID NO: 4, SEQ LD NO: 5, SEQ TD NO: 6, SEQ TD NO: 7, SEQ LD NO : 8, SEQ TD NO: 9, SEQ ID NO: 10 or SEQ TD NO: 11 or their homologue or derivative or fragment of functionally linked regulatory DNA sequence, e.g. B. a promoter, both the regulatory DNA sequence endogenously present with the nucleic acid sequence according to SEQ ID NO: 11, SEQ TD NO: 12, SEQ LD NO: 13, SEQ TD NO: 14, SEQ LD NO: 15, SEQ LD NO: 16, SEQ ID NO: 17, SEQ LD NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ HD NO: 21, SEQ LD NO: 22 or SEQ ID NO: 23 as well as their homologue or fragment as well as any other regulatory DNA sequence. The regulatory DNA sequence can be either an endogenous promoter of the organism to be transformed or an exogenous promoter which is e.g. B. is on the vector used. In principle, any regulatory sequence which expresses the expression of foreign genes in organisms, e.g. B. plants can control, e.g. B. the CaMV 35S promoter from the cauliflower mosaic virus; see. Franck et al., Cell 21: 285-294 (1980). The expression of the nucleic acid sequences can also be achieved by a chemically inducible promoter. Examples of chemically inducible promoters are the PRPI promoter (Ward et al., Plant Molecular Biology 22, 361-366 (1993)), a promoter induced by salicylic acid (WO 95/19443), a promoter inducible by benzenesulfonamide (EP-A 388186), a promoter inducible by tetracycline (Gatz et al, Plant Journal 2, 397-404 (1992)), a promoter inducible by abscisic acid (EP-A 335528) or a promoter inducible by ethanol or cyclohexanone (WO 93/21334) , Promoters can also be used, e.g. B. are active in certain plant tissues or parts of plants. Examples of corresponding promoters are the phaseolin promoter (US 5504200), the isoflavone reductase promoter (US 5750399), a seed-specific promoter, e.g. B. from tobacco (US 5824863) or the ST-LSI promoter from potato (Stockhaus et al., (1989), EMBO J8, 2445-2452). In this sense, the method according to the invention is suitable for time-controlled defense against pathogens by exchanging the WRKY-specific promoter for an inducible promoter. Through a time-controlled induction of the pathogen defense z. B. react to the seasonal occurrence of pathogens.

Endogenous or exogenous nucleic acid sequences according to SEQ LD NO: 1, SEQ TD NO: 2, SEQ TD NO: 3, SEQ ID NO: 4, SEQ LD NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ TD NO: 10 or SEQ ID NO: II or their fragments or derivatives or homologs can be used. Endogenous means that the nucleic acid sequence comes from the same organism in which it is integrated with the method according to the invention, e.g. B. a WRKY 33 gene from Arabidopsis thaliana is integrated into Arabidopsis thaliana using the method according to the invention. Exogenous means that the nucleic acid sequence comes from another organism, e.g. B. a WRKY 33 gene from Arabidopsis thaliana is with the inventive method in z. B. Wheat integrated. After the stable integration of the transformed nucleic acid sequence, two or more WRKY 33 genes are then present in the genome, so that an increased amount of corresponding gene products can be expected. In a preferred embodiment, the nucleic acid sequences have deletions, substitutions, additions, insertions and / or inversions compared to the naturally occurring nucleic acid sequences.

For the purposes of the invention, not only polynucleotides with the cDNA sequences of the WRKY genes according to SEQ ID NO: 1 to 11 or their fragments or derivatives or homologs can be used to produce the transgenic plants, but also polynucleotides which also contain the introns , The introns can have the naturally occurring or other sequences.

The present invention further relates to a transformed cell, in particular a transformed plant cell or a transformed plant tissue, in which the nucleic acid sequence according to the invention according to SEQ TD NO: 1, SEQ TD NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ TD NO: 6, SEQ TD NO: 7, SEQ TD NO: 8, SEQ TD NO: 9, SEQ LD NO: 10 or SEQ ID NO: II or their fragments or derivatives or homologs are stably integrated , Furthermore, the present invention relates to a nucleic acid sequence according to the invention according to SEQ TD NO: 1, SEQ LD NO: 2, SEQ LD NO: 3, SEQ LD NO: 4, SEQ LD NO: 5, SEQ ID NO: 6, SEQ LD NO: 7, SEQ TD NO: 8, SEQ ID NO: 9, SEQ TD NO: 10 or SEQ TD NO: 11 or their fragments or homologues or derivatives, transformed plant cell or a transformed plant tissue that can be regenerated into a fertile plant. In particular, the present invention relates to a plant which can be obtained by the process according to the invention. The present invention further relates to seed obtained from plants obtained by the process according to the invention. The invention further relates to the fruits, seeds, leaves, shoots and storage organs produced by the plants, e.g. B. fruits, berries, grapes, cereals and potatoes.

In a preferred embodiment of the method according to the invention, a nucleic acid sequence is used which is composed of nucleic acid fragments from different WRKY genes. This composite nucleic acid sequence can also contain modifications such as deletions, additions or substitutions. The methods for producing such composed nucleic acid sequences are state of the art and can be carried out easily by the person skilled in the art.

The method according to the invention can be applied to all plant organisms. In particular, the method according to the invention can be used for the production of pathogen-resistant mono- and dicotyledonous plants. Particularly preferred plants are cultivated plants such. B. coffee bush, tea bush, soybean, cotton, flax, hemp, sunflower, potato, tobacco, tomato, paprika, zucchini, eggplant, cucumber, summer rape, winter rape, alfalfa, lettuce, chicory, asparagus, salsify, pea, bean, lentils, Carrot, onion, garlic, leek, olive, radish, radish, beetroot, turnip, kohlrabi, sugar beet, fodder beet, sugar cane, spinach, chard, the different tree, nut and wine species, the different types of cabbage such as Brussels sprouts, cauliflower, broccoli, White cabbage, red cabbage, savoy cabbage, Chinese cabbage, and also cereals, e.g. B. barley, hops, wheat, rye, oats, corn, rice, feed grasses, and also fruit types such. B. mango, apple, pear, peach, plum, raspberry, blueberry, blackberry, gooseberry, strawberry, cherry, currant, guava, banana, melon, pumpkin and citrus z. As lemon, orange, grapefruit or tangerine and ornamental plants such. B. cyclamen, aster, bougainvillea, chrysanthemum, petunia, marguerite, narcissus, snowdrop, delphinium, sunflower, geranium, goose cress, gerbera, gladiolus, iris, hyacinth, lily, magnolia, orchid, rhododendron, rose, bleeding heart, tulip , finally all timber, needle and deciduous tree species such as B. Maple, birch, beech, yew, oak, ash, spruce, pine, linden, mahogany, poplar, fir, teak. The plants transformed with the nucleic acid sequence can be unmodified wild-type plants or plants obtained by breeding or plants which have already been modified in another way, e.g. B. be transgenic plants. The inventive method can also in plant tissues and in plant cells, for. B. in a cell culture or in expression systems, to increase pathogen resistance.

Since the pathogen defense based on WRKY proteins causes not only specific but also specific defense reactions, the method according to the invention can be used to improve the general resistance to a large number of plant pathogens. In particular, the method according to the invention can be used to protect against the following plant diseases, but is not limited to these: cabbage hernia, powdery scab of the potato, powdery mildew, downy mildew, late blight and late blight, powdery mildew, snow mold in the grain, ergot, root rot on rape, net stain disease , Septoria leaf drought, Septoria leaf spots and tan, brown rust, yellow rust, dwarf rust, wheat stone fire, dwarf stone fire, flying burn barley, corn bump fire, typhula rot on barley, root killers on potatoes, parasitic stalks, rhynchosporium leaf stains, leaf stains, cerciform spore, cerciform spore Alternaria leaf spot disease / rot, stem rot, core rot, leaf and stem rot, potato cancer, root burn, botrytis fruit rot, Colletotrichum fruit rot, strawberry powdery mildew, Gnomonia fruit rot, Phytophthora leather berry rot, Pseudomonas bacterial leaf blotch, Phseudomonas bacterial leaf blotch, Phytomonas bacterial leaf blotch, phyto-phytomorphic leaf blotch, phytophthalmic blotch phytomorphosis ia rot, red root rot, red spot disease, slime mold, verticillium wilt, soft rot of strawberry, white spot disease, cauliflower disease, angular leaf spot disease and all viroses.

Examples of corresponding pests which cause these and similar diseases and can be controlled by the method according to the invention are

I. Phytopathogenic nematode species, especially root nematodes, e.g. B. bile-forming 'root nematodes from the genus Meloidogyne, freely migrating

Root nematodes from the genera Pratylenchus and Paratylenchus, cyst-forming

Root nematodes from the genera Heterodera and Globodera; Stick and

Stem nematodes, e.g. B. from the genus Ditylenchus, further leaf nematodes, z. B. from the Genus Aphelenchoides and flower nematodes, e.g. B. from the genus Anguina, also virus-transmitting nematodes, for. B. from the genus Xiphinema and nematodes from the genus Trichodorus.

Relevant representatives of these genera, to which the method according to the invention can be applied, are e.g. B. Aphelenchoides ritzemabosi, fragariae, Aphelenchoides, Anguina gram ines, Anguina tritici, Globodera rostochiensis, Globodera pallida, Heterodera avenae, Heterodera cruciferae, Heterodera glycines, Heterodera schachtii, Heterodera rostochiensis, Meloidogyne javanica, Meloidogyne incognita, Pratylenchus zeae, Pratylenchus hexincisus, Pratylenchus penetrans, Pratylenchus fallax, Pratylenchus pseudocoffae, Pratylenchus coffae, Pratylenchus gutierrezi, Pratylenchus loosi, Pratylenchus neglectus, Pratylenchus vulnus, Pratylenchus thornei, Pratylenchus scribneri, Trichodorus primitivus, Xiphinema.

II. Phytopathogenic fungi, e.g. B. from the genera Albugo, Alternaria, Aphanomyces, Ascochyta, Aureobasidium, Botrytis, Cephalosporium, Cercospora, Cladosporium, Claviceps, Colletotrichum, Cylindrosporium, Diachea, Diplocarpon, Eiysiphe, Fusarium, Gnomporiumium, Macrominium, Macrominium, Macromiumium, Micomiumium, Helomium, Micomium, Micomium, Micomium, Micomium, Micomium, Helomium, Micomium, Micomium, Helomium, Micomium, Micomium, Helomium , Mycosphaefella, Oidium, Ophiobolus, Peronospora, Plasmodiophora, Plasmopora, Pseudocercosporella, Puccinia, Pyrenophora, Pythium, Phytophtora, Ramularia, Rhizoctonia, Rhynchosporium, Sclerophtora, Sclerhaopis, Sporia, Septoria, Septoria, Sporia , Venturia, Verticillium.

Relevant representatives of these genera, to which the method according to the invention can be applied, are e.g. B. Albugo candida, Alternaria alternata, Alternaria brassicae, Alternaria brassicicola, Alternaria solani, Aphanomyces cochlioides, Ascochyta hordei, Botrytis cinerea, Cephalosporium gramineum, Cercospora beticola, Claviceps purpurea, Colletotrichum trifoliorumaroparodirocarpus, cruciformia, cephalopods Erysiphe graminis, Fusarium ambrosium, Fusarium culmorum, Fusarium graminearum, Fusarium moniliforme, Fusarium oxysporum, Fusarium solani, Gnomonia fruticola, Helminthosporium avenae, Helminthosporium gramineum, Helminthosporium sativumporium Mucor piriformis, Mycosphaerella brassicola, Mycospaerella fragariae, Myrothecium roridum, Oidium cyclaminis, Oidium hortensiae, Oidium lycopersici, Oidium tuckeri, Ophiobolus graminis, Peronospora parasitica, Plasmodiophora brassicae, Plasmopora viticola, Puccinia hordei, Puccinia recondita, Puccinia coronata, Puccinia graminis, Puccinia striifomis, Phytophtora phactoraella pactoraella pactoraella pactoraella pactora pellora pellora phytocellella pactora pella porta Ramularia armoraciae, Ramularia beticola, Rhynchosporium secalis, Rhizoctonia cerealis, Rhizoctonia solani, Sclerophtora macrospora, Sclerotinia sclerotiorum, Septoria avenae, Septoria tritici, Septoria nodorum, Sphaeropsis sapinea, Sphaerhaercacais, sphaerothecais , Typhula gyrans, Typhula incarnata, Urocystis occulta, Ustilago avenae, Ustilago hordei, Ustilago maydis, Ustilago nuda, Ustilago tritici, Venturia inaequalis, Verticillium dahliae.

III Phytopathogenic bacteria, e.g. B. from the genera Agrobacterium, Erwinia, Pseudomonas, Ralstonia, Xanthomonas.

Relevant representatives of these genera, to which the method according to the invention can be applied, are e.g. B. Agrobacterium tumefaciens, Agrobacterium rhizobacter, Erwinia chrysanthemi, Pseudomonas syringae, Ralstonia solanacearum, Xanthomonas campestris.

IV. Phytopathogenic viruses, including single-stranded RNA viruses, viruses with double-stranded RNA (wound tumor viruses), plant viruses with circular single-stranded DNA (Gemini viruses), plant viruses with double-stranded DNA and viroids.

As typical representatives of phytopathogenic viruses z. B. be mentioned: abutilon mosaic virus, arabic mosaic virus, barley yellow dwarf virus (BYDV), barley yellow mosaic virus (BaYMV, BaYMV-2), bean golden mosaic virus, cassava latend virus, beet western yellows virus, cauliflower mosaic virus , grapevine fanleaf virus, maize streak virus, potato spindel tuber virus, raspberry ringspot virus, rice yellow mottle virus, strawberry crinkle virus, soil-borne wheat mosaic virus (SBWMV), strawberry yellow edge virus, strawberry mottle virus, tabac mosaic virus, tomato golden mosaic virus, turnip mosaic virus, wheat spindle streak mosaic virus (WSSMV), wheat yellow mosaic virus (WYMV).

V. Other animal pests in crops, z. B. Psylliodes chrysocephala, Ceutorhynchus pleurostigma, Ceutorhynchus napi, Ceutorhynchus quadridens, Ceutorhynchus picitaris, Ceutorhynchus assimilis, Melighetes aeneus, Delia brassicae, Dasineura brassicae and all types of insect pests in cereals.

The present invention further relates to the nucleic acid sequence according to SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ TD NO: 4, SEQ ID NO: 5, SEQ TD NO: 6, SEQ LD NO: 7, SEQ LD NO: 8, SEQ TD NO: 9, SEQ ID NO: 10 and SEQ TD NO: II or their homologue or derivative or fragment.

The present invention further relates to vectors comprising a nucleic acid sequence according to SEQ LD NO: 1, SEQ TD NO: 2, SEQ ID NO: 3, SEQ LD NO: 4, SEQ LD NO: 5, SEQ ID NO: 6, SEQ TD NO : 7, SEQ ID NO: 8, SEQ TD NO: 9, SEQ LD NO: 10 or SEQ ID NO: II or their homologue or derivative or fragment, and a regulatory DNA sequence.

Suitable vectors for the uptake and transfer of the nucleic acid sequences can multiply and / or express the uptake nucleic acids in single cells such as. B. Escherichia coli or Agrobacterium tumefaciens or in plant cells, plant tissues or plants. Corresponding vectors can of course occur or can be produced artificially. The vectors can include selection markers, terminator sequences, polylinkers, promoter elements, enhancers, polyadenylation sites and other genetic elements. Vectors suitable for cloning are e.g. B. plasmids of the pBluescript series, plasmids of the pUC series, plasmids of the pGEM series or vectors based on the bacteriophage λ. A plasmid vector used in Agrobacterium is e.g. B. pBinl9; see. Bevan et al., (1984), Nucleic Acids Research 12, 8711-8721. For transformation and expression in plants, vectors which can be used on the Ti plasmid of Agrobacterium species or constructs based on plant viruses are usable and are known to the person skilled in the art. Furthermore, certain transformation methods such. B. microinjection, protoplast transformation and injection (microprojectile bombardment) instead of Ti plasmids also the above-mentioned cloning rank vectors or linearized DNA used. A summary description of vectors used to date can be found in Guerineau and Mullineaux, Plant Transformation and Expression Vectors, in: Plant Molecular Biology Labfax, published by Croy, Oxford, BIOS Scientific Publishers, 121-148 (1993). Some of the transformation methods used in commercial transformation and expression systems for the transfer of foreign genes (transformation) into the genome of plants are presented below. However, the choice of the method for introducing the nucleic acid sequence according to the invention and the nucleic acid sequences functionally linked to it for a gene product into plant cells is not limited to this list. So far, transformation processes used in plants are e.g. B. the gene transfer by means of Agrobacterium tumefaciens (e.g. by bathing seeds, inflorescences or leaf fragments in an Agrobacterium solution), by means of plant viruses, by electroporation, by injecting (microprojectile bombardment) or injection (microinjection) and the incubation of dry embryos in DNA-containing Liquids and the transformation of protoplasts with the help of polyethylene glycol. More detailed descriptions of the methods mentioned can be found e.g. B. in Jens et al., Techniques for Gene Transfer, in: Transgenic Plants, Vol. 1, Engineering and Utilization, edited by Kung and Wu, Academic Press 128-143 (1993).

Another aspect of the present invention relates to the regulatory nucleic acid sequences (also referred to below as promoters) which naturally control the expression of the WRKY genes in Arabidopsis thaliana. These nucleic acid sequences according to the invention are under SEQ LD NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ LD NO: 15, SEQ LD NO: 16, SEQ LD NO: 17, SEQ LD NO: 18, SEQ LD NO : 19, SEQ TD NO: 20, SEQ LD NO: 21, SEQ LD NO: 22 and SEQ LD NO: 23.

The above SEQ TD NO: 12 to 23 can be found in the following list

Sequences described for registration for WRKY promoters can be assigned:

SEQ ID No: 12 corresponds to a 1270 bp fragment of the WRKY33 promoter.

SEQ TD No: 13 corresponds to a 361 bp fragment of the WRKY33 promoter.

SEQ TD No: 14 corresponds to the overall sequence of the WRKY33 promoter.

SEQ LD No: 15 corresponds to the overall sequence of the WRKY41 promoter. SEQ TD No: 16 corresponds to the overall sequence of the WRKY46 promoter.

SEQ LD No: 17 corresponds to the overall sequence of the WRKY54 promoter.

SEQ LD No: 18 corresponds to the overall sequence of the WRKY55 promoter.

SEQ LD No: 19 corresponds to the overall sequence of the WRKY62 promoter. SEQ LD No: 20 corresponds to the overall sequence of the WRKY63 promoter. SEQ LD No: 21 corresponds to the overall sequence of the WRKY64 promoter. SEQ ID No: 22 corresponds to the overall sequence of the WRKY67 promoter. SEQ TD No: 23 corresponds to the overall sequence of the WRKY70 promoter.

The invention further relates to fragments or homologs of the nucleic acid sequence according to SEQ LD NO: 12, SEQ LD NO: 13, SEQ LD NO: 14, SEQ TD NO: 15, SEQ TD NO: 16, SEQ ID NO: 17, SEQ TD NO: 18, SEQ LD NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ LD NO: 22 or SEQ ID NO: 23, which have the biological function of a promoter. The invention further relates to nucleic acid sequences which are associated with one of the nucleic acid sequences according to SEQ TD NO: 12, SEQ TD NO: 13, SEQ ID NO: 14, SEQ LD NO: 15, SEQ ID NO: 16, SEQ TD NO: 17, SEQ TD NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ LD NO: 21, SEQ TD NO: 22 or SEQ ID NO: 23 hybridize and have the biological form of a promoter. Preference is given to nucleic acid sequences which hybridize to one of these nucleic acid sequences under stringent conditions.

The regulatory nucleic acid sequences according to the invention according to SEQ ID NO: 12, SEQ TD NO: 13, SEQ TD NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ LD NO: 18, SEQ LD NO: 19, SEQ LD NO: 20, SEQ LD NO: 21, SEQ LD NO: 22 or SEQ J »NO: 23 or their fragments or homologs are suitable for the specific control of the expression of genes or other functional nucleic acids in organisms or cells , preferably for the specific control of genes or genes involved in pathogen defense, coding for toxic components. After the stable integration of the transformed nucleic acid sequence, two or more such promoters are then present in the genome, so that the downstream nucleic acid sequences have an expression regulation corresponding to these promoters.

On the basis of the immediate and local induction of the WRKY transcription factors shown here, by combining the regulatory nucleic acid sequences according to the invention according to SEQ ID NO: 12, SEQ ID NO: 13, SEQ TD NO: 14, SEQ ID NO: 15, SEQ TD NO: 16, SEQ LD NO: 17, SEQ TD NO: 18, SEQ TD NO: 19, SEQ TD NO: 20, SEQ ID NO: 21, SEQ ID NO: 22 or SEQ TD NO: 23 or their fragments or homologues with a toxic component or other genes which are suitable for defense against pathogens, for example, efficient broadband resistances are built up, the effect of which is limited to the site of infection. Thus, the pathogen can be combated without damaging the unaffected plant parts guaranteed.

The regulatory nucleic acid sequence according to the invention according to SEQ ID NO: 12, SEQ TD NO: 13, SEQ LD NO: 14, SEQ LD NO: 15, SEQ LD NO: 16, SEQ LD NO: 17, SEQ LD NO: 18, SEQ TD NO: 19, SEQ LD NO: 20, SEQ LD NO: 21, SEQ LD NO: 22 or SEQ LD NO: 23 or their fragment or homologously linked functional nucleic acids encode particularly preferably for gene products from the following list:

Structural proteins with repetitive peptide motifs Ser-Hyp ₄ , Gly-X or Val-Tyr-Lys-Pro-Pro. Structural proteins that contain repetitive peptide motifs with larger proportions of hydroxyproline (Hyp), proline (Pro) or glycine (Gly) can strengthen, change or repair the plant cell wall in the course of an infection and thus represent a barrier to the further penetration of the pathogen.

Hydrogen peroxide producing enzymes. Various proline-rich cell wall proteins are oxidatively linked to other proteins by H ₂ O ₂ after pathogen attacks and in this way strengthen the cell walls. Furthermore, aggressive oxygen derivatives in the context of the plant's hypersensitive immune response contribute to the death of the infected plant cells and help in the direct killing of the pathogen.

Lignin and callose producing enzymes. A frequently used defense strategy of the plant is the synthesis of polymeric cell wall components such as lignin or callose, which are built into the cell wall and serve as a mechanical barrier against the pathogen. An example of this class of substances is the "catalytic UE of callose synthase" (NCBI Acc. NO. AF085717).

Chitinases. Chitinases split the carbohydrate polymer chitin with the absorption of water. Chitin is an essential scaffold z. B. many phytopathogenic fungi. An example of this class of substances is "Chitinase 2" (NCBI Acc. NO. AF241267).

beta-l, 3-glucanases. ß-l, 3-glucanases cleave ß-l, 3-glucan with water absorption. This polymer also occurs as a scaffold in phytopathogenic fungi. Antipathogenically active enzyme inhibitors. The cell wall-bound polygalacturonidase inhibiting protein (PGLP), for example from the kidney bean Phaseolus vulgaris, can be mentioned here as an example. This inhibits the action of the enzyme α-1, 4-D-polygalacturonidase produced by the harmful fungus Fusarium monoliform, which the fungus uses to dissolve the bean cell walls.

Thionins. Thionins are small basic and cysteine-rich proteins that have so far been found mainly in cereals. An antifungal effect of thionines has been experimentally proven and is working on the ability of this class of proteins to destroy membranes.

Lectins with chitin-binding domains. Lectins are proteins with the ability to bind to carbohydrates in a highly specific manner. An antifungal lectin with a chitin-binding domain (NCBI Acc. NO. AAA34219) was isolated from nettle rhizomes.

Ribosome Inactivating Proteins (RIPs). Ribosome-inactivating proteins (see, for example, Leach et al., J. Biol. Chem. 266, 1564-1573, 1990) inhibit the protein synthesis of invading fungi and thus prevent their further growth. Plant-specific ribosomes are not damaged due to the different structure.

PR proteins ("Pathogenesis related"). The class of PR proteins (see, for example, Bol et al., Ann. Rev. Phytopathol. 28, 113-138, 1990) includes vegetable proteins found in the healthy plant not occur, but are synthesized after pathogen attack, examples include: PR-1 from Solanum tuberosum (NCBI Acc. NO. CAB58263), pathogenesis-related protein 4 from Hordeum vulgare (NCBI Acc. NO. T06171), transcription factor Pti6 from Solanum tuberosum (NCBI Acc. NO. T07728), thaumatin-like PR proteins, for example from Hordeum vulgare (NCBI Acc. NO. CAB99485).

Enzymes for the synthesis of phytoalexins. Phytoalexins are herbal secondary metabolites with strong antimicrobial activity that are accumulated around the site of infection. These include, for example, phenol derivatives, isoflavonoids and sesquiterpenes, e.g. B. chlorogenic acid, caffeic acid, scopoletin, Medicarpin, Glyceollin I, Rishitin, Gossypol, Lubimin, Capsidiol, Orchinol, Pisatin, Momilacton, Resveratol, Safynol. Enzymes for the synthesis of saponins. Saponins are steroid glycosides that can destroy fungal membranes by binding to sterols.

R gene-gene products. R genes (resistance genes) code for proteins that interact directly or indirectly via additional proteins with gene products from avr genes (aviralence genes) of the pathogen and lead to programmed cell death of infected plant cells at the infection site, e.g. B. NCBI Acc. NO. BE039015 (downy mildew resistance protein rpp5), NCBI Acc. NO. AF122994 (RPM1 variant gene, NCBI Acc. NO.AF098962 (disease resistance protein RPPl-WsA gene).

avr gene products. avr gene products are encoded by aviralence genes of the pathogen and activate the pathogen defense and the programmed cell death (apoptosis) of the plant cells at the site of infection in the course of an infection by contact with plant R-gene products.

Enzymes for the synthesis of salicylic acid. When a plant survives infection by a pathogen, it often develops resistance to a wide range of pathogens. This so-called "systemic acquired resistance (SAR) is mediated by the signaling molecule salicylic acid, which is first formed in the infection zone and later in various parts of the plant. The metabolic pathway leading to sahcylic acid synthesis starts from phenylalanine and is known to the person skilled in the art.

Enzymes for the synthesis of jasmonic acid. Jasmonic acid can be used to create resistance to Phytophtora infestans, for example, by applying the jasmonic acid externally. In the organism, jasmonic acid is injured when the plants, e.g. B. after pathogen contact, produces and activates various genes relevant for pathogen defense, z. B. proteinase inhibitors, thionins and plant defensin genes such. B. PDF1 (described in WO98 / 00023). Plant defensins are a family of cysteine-rich basic proteins with structural similarities to antimicrobial active defensins that have been detected in various insect species. The metabolic pathway underlying the synthesis of jasmonic acid starts from linolenic acid and is known to the person skilled in the art.

In a particularly preferred embodiment of the method according to the invention, further increase the efficiency of the pathogen defense by using several, ie two, three or many different gene promoter combinations, each consisting of a regulatory nucleic acid sequence according to the invention in accordance with SEQ LD NO: 12, SEQ LD NO: 13, SEQ LD NO: 14, SEQ LD NO: 15, SEQ LD NO: 16, SEQ LD NO: 17, SEQ LD NO: 18, SEQ TD NO: 19, SEQ TD NO: 20, SEQ TD NO: 21, SEQ TD NO: 22 or SEQ ID NO: 23 or its fragment or homolog and in each case a gene which is involved in the defense against pathogens or a gene which codes for toxic components according to the list above, can be integrated into the plant genome. For example, a combination 1 consisting of a regulatory nucleic acid sequence according to the invention and an R gene, together with a combination 2 consisting of a regulatory nucleic acid sequence according to the invention and an avr gene, can be integrated into the plant genome, so that by integrating both Gene-promoter combinations result in an increased activity against a pathogen.

The regulatory nucleic acid sequence according to the invention according to SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ TD NO: 15, SEQ LD NO: 16, SEQ TD NO: 17, SEQ TD NO: 18, SEQ LD NO: 19, SEQ LD NO: 20, SEQ LD NO: 21, SEQ LD NO: 22 or SEQ LD NO: 23 or their fragment or homolog functionally linked genes or other functional nucleic acids can endogenous or exogenous genomic DNA sections or cDNAs or their fragments or derivatives or synthetic or semisynthetic nucleic acids. Endogenous means that the nucleic acid sequence comes from the same organism in which it is integrated with the method according to the invention, e.g. B. a nucleic acid sequence from Arabidopsis thaliana is integrated into Arabidopsis thaliana using the method according to the invention. Exogenous means that the nucleic acid sequence comes from another organism, e.g. B. a nucleic acid sequence from Arabidopsis thaliana is with the inventive method in a crop, for. B. wheat, integrated. The functionally linked genes or other functional nucleic acid sequences can have deletions, substitutions, additions, insertions and / or inversions compared to the naturally occurring nucleic acid sequences.

The regulatory nucleic acid sequence according to the invention is also suitable for regulating the expression of other gene sequences. The promoter can be present in combination with any genes, both in a vector and in transgenic organisms. Nucleic acid sequences with an identical physiological function to the nucleic acid sequences according to SEQ LD NO: 12, SEQ ED NO: 13, SEQ TD NO: 14, SEQ TD NO: 15, SEQ ID NO: 16, SEQ LD NO: 17, SEQ LD NO: 18 , SEQ LD NO: 19, SEQ LD NO: 20, SEQ LD NO: 21, SEQ TD NO: 22 or SEQ ED NO: 23 can easily be extracted from other organisms with the help of special PCR, hybridization or Screening procedures are isolated. For example, the nucleic acid sequences listed above can be used as a probe for homology screening in DNA libraries with the aid of hybridization to single-stranded nucleic acids of a similar base sequence. By knowing the base sequence of the nucleic acid sequences listed above, primers can also be constricted, with the aid of which the amplification of PCR fragments coding for homologous gene products from other organisms is possible.

The regulatory nucleic acid sequences according to the invention according to SEQ TD NO: 12, SEQ ID NO: 13, SEQ LD NO: 14, SEQ ID NO: 15, SEQ ED NO: 16, SEQ ID NO: 17, SEQ LD NO: 18, SEQ ID NO : 19, SEQ ID NO: 20, SEQ TD NO: 21, SEQ TD NO: 22 or SEQ ED NO: 23 or their fragments or homologues can be of natural origin or have been produced artificially. The genes which are functionally connected thereto or the other functional nucleic acid sequences can be present both in the sense rank which gives the natural direction of transcription and in the reverse antisense orientation.

The regulatory nucleic acid sequence according to the invention according to SEQ TD NO: 12, SEQ TD NO: 13, SEQ TD NO: 14, SEQ ID NO: 15, SEQ ED NO: 16, SEQ TD NO: 17, SEQ ED NO: 18, SEQ ED NO: 19, SEQ ID NO: 20, SEQ ED NO: 21, SEQ ED NO: 22 or SEQ ED NO: 23 or their fragments or homologs can be modified in vectors, expression systems or plants, plant tissues or plant cells or animal cells or microorganisms the expression pattern of various gene products can be used. The expression of the gene products can be both increased and reduced compared to their natural expression.

The present invention accordingly further relates to vectors comprising a regulatory nucleic acid sequence according to SEQ LD NO: 12, SEQ TD NO: 13, SEQ TD NO: 14, SEQ LD NO: 15, SEQ LD NO: 16, SEQ LD NO: 17, SEQ TD NO: 18, SEQ ED NO: 19, SEQ LD NO: 20, SEQ LD NO: 21, SEQ ED NO: 22 or SEQ ED NO: 23 or their fragment or homolog.

Suitable vectors for recording and transferring the nucleic acid sequences and also some of the transformation methods used in commercial transformation and expression systems for the transfer of foreign genes (transformation) into the genome of plants have already been mentioned above.

The present invention further relates to a method for producing a plant with modified gene expression, comprising the stable integration of a regulatory sequence according to SEQ LD NO: 12, SEQ TD NO: 13, SEQ TD NO: 14, SEQ TD NO: 15, SEQ ED NO : 16, SEQ TD NO: 17, SEQ ED NO: 18, SEQ ED NO: 19, SEQ TD NO: 20, SEQ ED NO: 21, SEQ ED NO: 22 or SEQ ED NO: 23 or their fragment or homologue with the biological function of a promoter and a nucleic acid sequence which is functionally linked to a gene product and codes for a gene product in the genome of plant cells or plant tissues and regeneration of the plant cells or plant tissues obtained to give plants.

Endogenous or exogenous nucleic acid sequences according to SEQ TD NO: 12, SEQ TD NO: 13, SEQ TD NO: 14, SEQ TD NO: 15, SEQ TD NO: 16, SEQ HD NO: 17, SEQ ID NO: 18, SEQ TD NO: 19, SEQ ID NO: 20, SEQ ED NO: 21, SEQ ED NO: 22 or SEQ ED NO: 23 or their fragments or homologs can be used. After the stable integration of the transformed nucleic acid sequence, two or more WRKY 33 promoters are then present in the genome, so that several genes are regulated by these promoters. In a preferred embodiment, the nucleic acid sequences have deletions, substitutions, additions, insertions and / or inversions compared to the naturally occurring nucleic acid sequences.

The method according to the invention can be applied to all plant organisms. In particular, the method according to the invention can be used for the production of pathogen-resistant mono- and dicotyledonous plants. Particularly preferred plants are the crop plants already listed above.

The invention further relates to transgenic plants with a regulatory nucleic acid sequence stably integrated into the genome according to SEQ LD NO: 12, SEQ TD NO: 13, SEQ ED NO: 14, SEQ ED NO: 15, SEQ ID NO: 16, SEQ TD NO: 17, SEQ TD NO: 18, SEQ ID NO: 19, SEQ TD NO: 20, SEQ ID NO: 21, SEQ LD NO: 22 or SEQ ID NO: 23 or its fragment or homolog with the biological function of a promoter and a nucleic acid sequence which is functionally linked to a nucleic acid sequence and which codes for a gene product, corresponding to the examples of such coding nucleic acid sequences listed and described above.

The present invention further relates to a transformed cell, in particular a transformed plant cell or a transformed plant tissue, in which the nucleic acid sequence according to the invention according to SEQ LD NO: 12, SEQ ED NO: 13, SEQ ID NO: 14, SEQ TD NO: 15, SEQ TD NO: 16, SEQ TD NO: 17, SEQ ED NO: 18, SEQ TD NO: 19, SEQ HD NO: 20, SEQ TD NO: 21, SEQ TD NO: 22 or SEQ ID NO: 23 or their fragments or homologs , are stably integrated. The present invention further relates to a nucleic acid sequence according to the invention according to SEQ TD NO: 12, SEQ ED NO: 13, SEQ ID NO: 14, SEQ ED NO: 15, SEQ ED NO: 16, SEQ ED NO: 17, SEQ ED NO : 18, SEQ ED NO: 19, SEQ HD NO: 20, SEQ ED NO: 21, SEQ ED NO: 22 or SEQ ED NO: 23 or their fragments or homologues transformed plant cell or a transformed plant tissue, or a transformed plant tissue, which or a fertile Plant is regenerable. In particular, the present invention relates to a plant which can be obtained by the process according to the invention. The present invention further relates to seed obtained from plants obtained by the process according to the invention. The invention further relates to the fruits, seeds, leaves, shoots and storage organs produced by the plants, e.g. B. fruits, berries, grapes, cereals and potatoes.

The plants transformed with the nucleic acid sequence can be unmodified wild type plants or plants obtained by breeding or modified plants e.g. B. be transgenic plants. The inventive method can also in plant tissues and plant cells, for. B. in a cell culture or in expression systems, to increase pathogen resistance.

Since the pathogen defense based on WRKY proteins causes not only specific but also specific defense reactions, the method according to the invention can be used to improve the general resistance to a large number of plant pathogens. In particular, this method according to the invention can also be used to protect against the plant diseases and pests already described above (nematodes, fungi, bacteria, viruses and other animal pathogens of the types already mentioned above), but is not limited to these.

The invention is illustrated by the following examples, but is not limited to these:

Example 1: Quantitative RT-PCR To identify WRKY genes which are induced during the defense of the Arabidopsis plants against Peronospora parasitica, a quantitative RT-PCR approach was chosen and the expression of specific WRKY genes was checked.

First the pathogen Peronospora parasitica pv Cala was grown on Ler-1 (Arabidopsis thaliana var. Landsberg erecta) plants and the sporangiophores and spores were harvested by cutting off the leaves and cotyledons of the Ler-1 plants. To get the spores in solution, the leaves and cotyledons were shaken in sterile-filtered, double-distilled water. The spores were pelleted by centrifugation at 4000 × g at 10 ° C. for five minutes and taken up in new, double-distilled water. A spore solution of 2x10 ⁵ spores / ml was counted and set under the microscope using a counting chamber. The spores were then sprayed onto two-week-old plants grown under short day. Plants were harvested at about 1 to 2 hour intervals and frozen in liquid nitrogen. This plant material was used for RNA isolation. The Quiagen-RNA / DNA-Maxi-Kit was used for this RNA isolation system and was operated exactly according to the manufacturer's instructions. For the RT-PCR approach, RNA quantities between 10 pg and 2 μg were used. The concentration of the RNA was determined photometrically at 260 nm. In a first run, different amounts of RNA were used in the RT-PCR approaches in order to determine the optimal concentration to be used for a specific WRKY-RNA. This ensured that the amount of cDNA obtained in the RT-PCR was still in the exponential and not already in the saturated range. The RT-PCR batches were all carried out using the one-step RT-PCR kit (Qiagen). The manufacturer's instructions were followed closely. The program in the thermal cycler (model PTC225 from MJResearch) was entered as follows: 30 min 50 ° C; 15 min 95 ° C; 35 cycles with 40 sec 94 ° C, 1 min 55 ° C or 65 ° C depending on the primer sequence (AfWK55-3 and AtWK55- 6: 65 ° C; all other primers: 55 ° C), 1 min 72 ° C; followed by 10 min 72 ° C and then 4 ° C until the batches are removed. The following primers were used (sequence from 5 'to 3'):

For RKY30 AtWRKY30-3: TCGAAGAAGTCAATGCCAAGGTGGAG (SEQ ID NO: 35) AtWRKY30-4: TTTGATGCTGAGTGAGAAGCCGAGCC (SEQ ID NO: 36)

For WRKY41 At RKY41-lb: ATGGAAATGATGAATTGGGAGCGGAGG (SEQ ID NO: 37) AtWRKY41-5: GCCTGTGTTAATCTCAGCCGTTGGATC (SEQ ID NO: 38)

For RKY46 AtWRKY46-l: GGAAGGTATCGGAGAAGAACACACAGAG (SEQ ID NO: 39) At RKY4β-2: CACCGTTCTCAATCTCATGGTTAGAG (SEQ ID NO: 40)

For RKY54 AtWRKY54-2: GCTTCCACGATCCTTGTATGTGATCT (SEQ ID NO: 41) AtWRKY54-3: ATGGATTCGAATAGTAACAACACGAAATCC (SEQ ID NO:

42)

For RKY55 At RKY55-3: AAATCGCTCGAGTCCAGCTTACCGGA (SEQ ID NO: 43) At RKY55-6: CATGTTGGTTCCGACCCCGCCGCTAC (SEQ ID NO: 44)

For WRKY62 At RKY62-3: GTTTCCAACATTGATCACAAGGCTATG (SEQ ID NO: 45) At RKY62-4: ACATTCTGTCACTCGATGGAAACGGG (SEQ ID NO: 46)

For RKY63 At RKY63-3: AACATCGATCACAAGGCTGTGGCAGC (SEQ ID NO: 47) At RKY63-4: CAAAACAACATCAGGTCTTCCGATGA (SEQ ID NO: 48)

For WRKY64 AtWRKY64-3: CTCCAACATCGATCAAACAGCTGTGG (SEQ ID NO: 49) AÜWRKY64-4: TGATATGGTCTATGGCTTCGTCCTCC (SEQ ID NO: 50)

For RKY67 At RKY67-l: AAGATGAACTCTTGCCAACAAAAGGC (SEQ ID NO: 51) At RKY67-3: CATGATGATAAGTCGTGAGATGTCCAG (SEQ ID NO: 52)

For WRKY70 At RKY70-lb: GCTTAAAGTTATGAACCAACTCGTTG (SEQ ID NO: 53) At RKY70-2: TCATGGTCTTAGTCCTATAGTAGTGG (SEQ ID NO: 54)

The cDNA obtained was separated on a 0.8% agarose gel in TAE and photographed. Analysis of some Arabidopsis WRKY genes showed that they are induced during the defense reaction of the Arabidopsis thaliana Col-0 plants against Peronospora parasitica pv. Cala2. Other WRKY genes were not induced. The WRKY genes induced were the following: WRKY 30, WRKY33, WRKY 41, WRKY46, WRKY 54, WRKY 55, WRKY 62, WRKY 63, WRKY 64, WRKY 67, WRKY 70.

Example 2: Northern analysis 1) Northern analyzes were carried out to further demonstrate the induction of WRKY33 by the infestation of Peronospora parasitica. For this purpose, RNA was isolated from Arabidopsis plants (ecotype Col-0) sprayed with Peronospora parasitica pv.Cala. The Quiagen-RNA / DNA-Maxi-Kit was used for this RNA isolation and proceeded exactly according to the manufacturer's instructions. 20 μg RNA per lane were applied to a formamide agarose gel and electrophoresed overnight using a voltage of 1 volt per cm electrode spacing. The RNA was then blotted onto a PALL-B membrane (Pall Corp., New York, USA), whereby the manufacturer's instructions were also followed exactly. 20xSSC (175.3 g / 1 NaCl, 88.2 g / 1 sodium citrate, pH 7.0) was selected as the transfer buffer and blotted capillary for 24 hours. The RNA was then heat-fixed to the membrane by baking the membrane at 80 ° C for 3 hours. In order to show the expression of the WRKY33 gene, the membrane was hybridized with a WRKY 33 specific probe. For this purpose, a 350 bp long fragment was amplified by PCR and this was labeled with radioactive P32-dCTP after elution with the QuiaexII gel extraction kit manufactured by Quiagen (method according to the manufacturer). The Ready-To-Go ™ kit from Amersham Pharmacia Biotech Inc. was used for the labeling reaction. The hybridization took place overnight in 12% o dextran sulfate buffer (1 M NaCl; 1% SDS; 12% dextran sulfate) at 65 ° C and was washed stringently with 2xSSC / 0.5% SSC at 62 ° C. It was then determined by means of autoradiography whether there was an induction of the expression of WRKY 33 in the Arabidopsis plants sprayed with the avirulent Peronospora strain Cala2. A clear signal in the case of plants sprayed with Peronospora parasitica pv Cala2 was detectable as early as 2 hours after contact with the pathogen. In contrast, no signal was detectable in Arabidopsis plants which were only sprayed with water. It follows that WRKY33 is induced in the defense reaction of the Col-0 plants against Peronospora parasitica Cala2.

2) Further Northern Bio t analyzes should clarify where WRKY33 is in the signal chain. The Northern blots were carried out as described in Example 2.1). In transgenic plants that contain a bacterial nahG construct that leads to the degradation of salicylic acid (SA), the signal chain is therefore inhibited from this level. The expression of WRKY33 is not affected by this and therefore WRKY33 is obviously above the salicylic acid in the signal chain. The analysis of pad4-l mutants (in the Col-0 ecotype) shows a strong infection and a significant delay after Perosnospora parasitica infections Expression of WRKY33 from 4 hours after infection (hpi = hours post induction) to 72 hpi. An identical delay was also found after infection of Ler-1 (Arabidopsis thaliana var. Landsberg erecta) plants with Perosnospora parasitica pv Cala2 for WRKY33. These results indicate that although WRKY33 is also induced in the susceptible interaction between Ler-1 and Perosnospora parasitica pv Calal, the time of induction is so late that it can be assumed that WRKY33 has a specific role in signal transduction in the resistant pathogen defense plays. WRKY33 also plays a role in RPP-4-dependent signal transduction, since it is also expressed very early (2-4 hpi) after an Emoy2 infection on Col-0.

3) Northern analysis for the detection of "immediate early genes"

To demonstrate that WRKY33 belongs to the "immediate early" genes, an Arabidopsis cell culture was shaken for 30 minutes with cycloheximide (final concentration 25 μM). RNA was then isolated from this cell culture. For this RNA isolation, the Quiagen RNA / DNA maxi kit used and proceeded exactly according to the manufacturer's instructions. A Northern analysis was carried out as described in Example 2.1). Cycloheximide stops protein biosynthesis, so that only "immediate early" genes are expressed. Since a very clear signal was visible in the autoradiography in this Northern analysis, WRKY 33 can be classified as “immediate early” genes.

Example 3: Promoter-CIS Contracts

In order to determine whether other pathogens also influence WRKY gene expression, promoter-GUS constructs were produced and stably integrated into the genome of Arabidopsis plants. In this way it could be shown that very different pathogens, e.g. B. Peronospora, Pythium, Altemaria and Sclerotinia, which induce expression of WRKY33. In addition, it could be shown that the induction of this gene is locally limited and is located directly around the infection site. To determine which sequence is that of the promoter, the start of transcription was identified. S'Race experiments were carried out to determine the transcription start point of the WRKY33 gene. The Boehringer 5'-3'RACE kit was used and the manufacturer's instructions followed. After the transcription start point had been determined, promoter regions of different lengths were amplified using PCR and gene-specific primers as shown in Figure 21. The following were used as forward primers:

For the 1270 bp fragment: 5'-atgctaagcttgccaaagggtgttgttatt-3 '(SEQ TD NO: 55)

For the 361 bp fragment: 5 '-aattaagcttgagagtcaaggccacaaggt-3' (SEQ ED NO: 56)

For the 331 bp fragment: 5'-atgttaagcttctatctcttctccttcttc-3 '(SEQ TD NO: 57) For the 193 bp fragment: 5'-atgttaagcttcagacgtgcaat-3' (SEQ HD NO: 58)

The recognition sequences for a restriction enzyme are underlined in each case. The reverse primer for all four fragments mentioned above was used: 5'-ccggggatccggttctgctattgtccattgtaag-3 '(SEQ HD NO: 59)

The program in the thermal cycler (model PTC225 from MJResearch) was entered as follows: 150 seconds at 94 ° C, 60 seconds at 80 ° C (addition of Tfu polymerase, HotStart method), 38 cycles with 60 seconds at 60 ° C, 100 7 sec PC, 45 sec 94 ° C, followed by 60 sec 60 ° C and 90 sec 71 ° C.

The amphfected promoter fragments were again cloned by means of PCR using the Tfu polymerase (Stratagene) in front of a GUS reporter gene, so that continuous translation was possible. The constructs were placed in the pGPTV vector (Plant Mol Biol. 20 (6), 1195-1197 (1992)) and stably integrated into the genome of Arabidopsis plants ecotype Col-0 by means of Agrobacterium transformation. The transformants were identified using selection plates. The vector used contains kanamycin resistance and thus made it possible to select transformants on kanamycin-containing plates. After selection, these plants were transplanted to soil and grown. The seeds of these plants were used for the application of various pathogens. For this purpose, the seeds were applied sterile on MS plates or grown directly on earth. The pathogens were administered in accordance with the following specialist literature: (Pythium: Vijayan, P. et al.: A role for jasmonate in pathogen defense of Arabidopsis. Proc. Nat. Acad. Sci.USA 95, 7209-7214, 1998. Altemaria and Sclerotinia as described in the following publication for Rhizoctonia: Broekaert et al: An automated quantitative assay for fungal growth inhibition FEMS Microbiology Letters 69, 55-60, 1990). All the pathogens tested showed a clear and local induction of the WRKY33 gene. claims

1. Transgenic plant with improved resistance to pathogens, the improved resistance by an increased expression of the nucleic acid sequence according to SEQ HD NO: 2, SEQ HD NO: 6, SEQ HD NO: 5, SEQ HD NO: II, SEQHD NO: 3, SEQ TD NO: 7, SEQ HD NO: 8, SEQ ID NO: 4, SEQ TD NO: 10, SEQ HD NO: 1 and / or SEQ HD NO: 9, or their homologue or derivative or fragment, or by the change the biological activity of the nucleic acid sequence according to SEQ HD NO: 2, SEQ HD NO: 6, SEQ HD NO: 5, SEQ HD NO: 11, SEQ ID NO: 3, SEQ ID NO: 7, SEQ ID NO: 8, SEQ TD

NO: 4, SEQ ID NO: 10, SEQ ED NO: 1 and / or SEQ HD NO: 9, or their homologue or derivative or fragment encoded gene product.

2. Transgenic plant according spoke 1 with increased expression of the nucleic acid sequence according to SEQ HD NO: 2, SEQ HD NO: 6, SEQ HD NO: 5, SEQ HD NO: 11, SEQ ED NO: 3, SEQ HD

NO: 7, SEQ HD NO: 8, SEQ HD NO: 4, SEQ HD NO: 10, SEQ ED NO: 1 and or SEQ HD NO: 9, or their homologue or derivative or fragment, the nucleic acid sequence according to SEQ HD NO : 2, SEQ HD NO: 6, SEQ HD NO: 5, SEQ HD NO: ll, SEQ TD NO: 3, SEQ TD NO: 7, SEQ ID NO: 8, SEQ ED NO: 4, SEQ HD NO: 10 , SEQ TD NO: 1 and / or SEQ HD NO: 9, or their homologue or derivative or fragment and one with this (these)

Nucleic acid sequence (s) functionally linked regulatory nucleic acid sequence is (are) also stably integrated into the genome.

3. Transgenic plant according to spoke 2, comprising at least two copies of one of the nucleic acid sequences according to SEQ HD NO: 2, SEQ TD NO: 6, SEQ ID NO: 5, SEQ TD

NO: II, SEQ TD NO: 3, SEQ ED NO: 7, SEQ ID NO: 8, SEQ HD NO: 4, SEQ HD NO: 10, SEQ HD NO: 1 and / or SEQ ED NO: 9, or their Homologue or derivative or fragment.

4. Transgenic plant according to spoke 2 or 3, wherein the regulatory nucleic acid sequence is selected from the group of promoters CaMV 35S promoter, ubiquitin promoter,

PRPI promoter, phaseolin promoter, isoflavone reductase promoter, ST-LSI promoter, salicylic acid inducible promoter, benzenesulfonamide inducible promoter, tetracycline inducible promoter, abscisic acid inducible promoter, ethanol or cyclohexanone inducible promoter, promoter according to any one of claims 10 to 12, or its fragment or homologue with the biological function of a promoter, or a seed-specific tobacco promoter.

5. Transgenic plant according to claim 1 with increased expression of the nucleic acid sequence according to SEQ HD NO: 2, SEQ LD NO: 6, SEQ HD NO: 5, SEQ HD NO: 11, SEQ ID NO: 3, SEQ ID NO: 7, SEQ HD NO: 8, SEQ HD NO: 4, SEQ HD NO: 10, SEQ HD NO: 1 and or SEQ HD NO: 9, or their homolog or derivative, the endogenous promoter of the nucleic acid sequence according to SEQ HD NO: 2, SEQ HD NO: 6, SEQ HD NO: 5, SEQ TD NO: 11, SEQ HD NO: 3, SEQ HD NO: 7, SEQ HD NO: 8, SEQ HD NO: 4, SEQ TD NO: 10, SEQ TD NO : l and / or SEQ ID NO: 9, or their homolog or derivative, is modified by changing or adding functional components.

6. Transgenic plant according spoke 1 with increased expression of the nucleic acid sequence according to SEQ HD NO: 2, SEQ HD NO: 6, SEQ TD NO: 5, SEQ HD NO: l 1, SEQ HD NO: 3, SEQ HD

NO: 7, SEQ HD NO: 8, SEQ HD NO: 4, SEQ ED NO: 10, SEQ ED NO: 1 and / or SEQ ED NO: 9, or their homolog or derivative, the endogenous promoter of the nucleic acid sequence according to SEQ LD NO: 2, SEQ HD NO: 6, SEQ HD NO: 5, SEQ HD NO: 11, SEQ HD NO: 3, SEQ TD NO: 7, SEQ HD NO: 8, SEQ HD NO: 4, SEQ ED NO : 10, SEQ HD NO: 1 and / or SEQ ED NO: 9, or their homolog or derivative, is replaced by a strong promoter, in particular by a CaMV 35S promoter from the cauliflower mosaic virus or the ubiquitin promoter Corn.

7. Transgenic plant according spoke 1, wherein the changed biological activity of the by the nucleic acid sequence according to SEQ ED NO: 2, SEQ HD NO: 6, SEQ ID NO: 5, SEQ ID

NO: II, SEQ HD NO: 3, SEQ HD NO: 7, SEQ HD NO: 8, SEQ HD NO: 4, SEQ ED NO: 10, SEQ HD NO: 1 and / or SEQ HD NO: 9 or their homologue or derivative or fragment of encoded gene product by modifying functional protein domains, in particular receptor binding, phosphoryl and / or DNA binding domains, in particular the WRKY DNA binding domain.

8. The transgenic plant of claim 7, wherein the modifications include deletions, additions or substitutions of amino acid residues. 9. Nucleic acid sequence according to SEQ HD NO: 2, SEQ HD NO: 6, SEQ HD NO: 5, SEQ HD NO: 11, SEQ HD NO: 3, SEQ HD NO: 7, SEQ HD NO: 8, SEQ HD NO: 4, SEQ HD NO: 10, SEQ HD NO: 1 and / or SEQ HD NO: 9, or their homologue or derivative or fragment.

10. Nucleic acid sequence according to SEQ HD NO: 12, SEQ TD NO: 13, SEQ TD NO: 14, SEQ TD NO: 18, SEQ TD NO: 17, SEQ TD NO: 23, SEQ TD NO: 15, SEQ HD NO: 19, SEQ ID NO: 20, SEQ TD NO: 16, SEQ TD NO: 22 or SEQ ID NO: 21.

11. Fragment or derivative of the nucleic acid sequence according to spoke 10 or a nucleic acid sequence that corresponds to the nucleic acid sequence according to SEQ HD NO: 12, SEQ ID NO: 13, SEQ TD NO: 14, SEQ TD NO: 18, SEQ ID NO: 17, SEQ HD NO: 23, SEQ HD NO: 15, SEQ HD NO: 19, SEQ ID NO: 20, SEQ HD NO: 16, SEQ HD NO: 22 or SEQ ID NO: 21 and has the biological activity of a promoter.

12. Nucleic acid sequence according to spoke 11, wherein the hybridizing nucleic acid sequence under stringent conditions with the nucleic acid sequence according to SEQ LD NO: 12, SEQ HD NO: 13, SEQ HD NO: 14, SEQ ED NO: 18, SEQ HD NO: 17, SEQ HD NO: 23, SEQ ED NO: 15, SEQ ED NO: 19, SEQ HD NO: 20, SEQ HD NO: 16, SEQ TD NO: 22 or SEQ ED NO: 21 hybridized.

13. Transgenic plant with at least one regulatory nucleic acid sequence stably integrated into the genome after its transformation and according to one of claims 10 to 12 and a nucleic acid sequence which is functionally linked to a nucleic acid sequence and codes for a gene product.

14. The transgenic plant according to spoke 13, wherein the nucleic acid coding for a gene product is selected from the nucleic acids coding for starch proteins with repetitive peptide motifs Ser-Hyp ₄ , Gly-X or Val-Tyr-Lys-Pro-Pro, H ₂ θ ₂ -producing enzymes, lignin and callose producing enzymes, chitinases, ß-l, 3-glucanases, antipathogenically active enzyme inhibitors, thionines, lectins with chitin-binding domains, ribosome-inactivating proteins, PR proteins, enzymes for the synthesis of phytoalexins, enzymes for the synthesis of saponins, R gene gene products, avr- Gene products, enzymes for the synthesis of salicylic acid.

15. The transgenic plant according to claim 13 or 14, wherein two or more different combinations of regulatory nucleic acid sequences and nucleic acid sequences coding for a gene product are stably integrated into the genome.

16. A method for producing a transgenic plant according to any one of claims 1 to 6 or 13 to 15, comprising the stable integration of a regulatory nucleic acid sequence, in particular a nucleic acid sequence according to one of claims 10 to 12, and optionally a functionally linked to this nucleic acid sequence for a

Nucleic acid sequence coding gene product, in particular the nucleic acid sequence according to claim 9, into the genome of plant cells or plant tissues and regeneration of the plant cells or plant tissues obtained to plants.

17. Transformed plant cell or transformed plant tissue with at least one regulatory nucleic acid sequence stably integrated into the genome after its transformation, in particular a nucleic acid sequence according to one of claims 10 to 12 and optionally a nucleic acid sequence coding for a gene product, in particular the nucleic acid sequence according to speech, which is functionally linked to this nucleic acid sequence 9th

18. Plant cell or plant tissue according to spoke 17, regenerable to a fertile plant.

19. Seed obtained from plants according to one of claims 1 to 8 or 13 to 15.

20. Vector comprising a nucleic acid sequence according to one of claims 9 to 12.

21. A method for producing transgenic plants using the vector of claim 20.